In preliminary studies we have shown that Cd++ is unique among the heavy metals: It is a very potent synaptic blocking agent but, unlike Pb++ or La+++, has little affect on presynaptic Ca++ uptake and it decreases MEPP frequency. These studies indicate that Pb++ inhibits transmitter release by competing with Ca++ at outer membrane sites, thereby reducing inward Ca++ flux and Cd++ by competing with Ca++ at intracellular sites, thereby preventing vesicle-membrane fusion. Our general objective is to clarify the mechanisms and sites of actions of Ca++ in transmitter release using Cd++, Pb++ and other divalent cations as tools. The following experiments are proposed: I. Electrophysiology experiments using the frog sciatic nerve-sartorius muscle preparation will quantify the effects of Cd++ on evoked and spontaneous transmitter release and compare these effects to those of divalent cations known to reduce inward Ca++ conductance: (a) determination of whether Cd++ is a competitive inhibitor of Ca++ in evoked transmitter release (b) study of the combined effects of Cd++ + Pb++ or Cd++ + Mg++ on the EPP: The kinetic relationships between EPP and metal concentration should indicate whether one or two receptor sites are involved (c) quantitative characterization of the effects of Cd++ on MEPP frequency. II. Biochemical experiments using Torpedo electroplaque and bullfrog sympathetic ganglion will examine the effect of Cd++ and other metals on Ca++ fluxes in nerve terminals and the release of ACh, and identify binding sites at which Cd++ and Pb++ compete with Ca++: (a) 45Ca uptake of nerve terminals of frog sympathetic ganglion using Pb++, Cd++, Co++ and Mg++. (b) Using Torpedo synaptosomes the kinetics of 45Ca influx and efflux will be determined, then the effects of Cd++ and Pb++ assessed. (c) Inhibition by Cd++ and Pb++ of K-stimulated ACh release from Torpedo synaptosomes. (d) ACh release from synaptosomes treated with Ca++ ionophore A-23187 will be examined: Cd++ but not Pb++ should reduce the ACh release. (e) Electroplaque synaptosomes will be used to determine whether Cd++ enters nerve terminal via the Ca++ channels. (f) Interaction of Ca++, Pb++ and Cd++ with synaptic vesicles and synaptic membranes of electroplaque will be examined to identify binding sites on the outer and inner synaptic membranes and vesicular membranes.